Phospholipase Cgamma Pathway-Dependent mTOR Signaling Controls IL-7-Mediated Early B Cell Development

Early B cell lymphopoiesis is intricately regulated by signals from various receptors including the pre-B cell receptor (pre-BCR) and the interleukin-7 receptor (IL-7R), but the molecular mechanism underlying the regulation is not fully understood. The Phospholipase Cγ (PLCγ) signaling pathway plays a critical role in antigen receptor-mediated activation of mature B cells, but the role of this pathway in cytokine function and early B cell development is completely unknown. Here we generated inducible PLCγ1/PLCγ2 double-deficient mice and found that the mutant mice exhibited a complete blockage of early B cell development at the pre-pro-B cell stage, similar to IL-7R-deficient but not pre-BCR-deficient mice. Of note, PLCγ1/PLCγ2 double-deficient mice had normal numbers of the HSC-containing Lin^-c-Kit⁺Sca1⁺ (LSK) cells, myeloid progenitor-containing Lin^-c-Kit⁺Sca1^- (LK) cells and myeloid lineage cells compared to wild-type mice. PLCγ1/PLCγ2 double-deficient mice displayed a marked reduction in IL-7-induced rearrangement of the distal V_HJ558 family gene segments. In addition, PLCγ1/PLCγ2 double deficiency altered the expression of lymphoid-lineage-affiliated genes in common lymphoid progenitors (CLPs) and the expression of B cell lineage-affiliated genes, IL-7 signaling pathway-regulated genes and cell cycle pathway-related genes in B cell progenitors. Further, PLCγ1/PLCγ2 double-deficient Lin^- BM progenitors co-cultured with OP-9 stromal cells plus IL-7 failed to give rise to B cells whereas wild-type control BM progenitors generated B cells. PLCγ1/PLCγ2 double-deficient B progenitors exhibited a decrease in the level of IL-7-induced BrdU uptake compared to the corresponding wild-type control cells. Biochemically, the PLCγ pathway was activated upon IL-7 engagement in Rag1-deficient pro-B cells that lack the pre-BCR. PLCγ1/PLCγ2-double deficiency or inhibition of PLCγ pathway by PLC inhibitors impaired IL-7-induced activation of mTORC1 but not Stat5. Consistently, inhibition of the PLCγ pathway impaired IL-7-mediated metabolism of B cell progenitors. It's known that PLCγ activates the DAG/PKC-dependent and IP₃/Ca²⁺-dependent signaling pathways, the two major downstream signaling cascades. We found that inhibition of the PKC-dependent, but not the Ca²⁺-dependent, pathway impaired IL-7-mediated B cell production and mTORC1 but not Stat5 activation. In addition, the PKC agonist PDBu induced mTORC1 activation in wild-type B cell progenitors, and stimulated ³H-thymidine incorporation in PLCγ1/PLCγ2 double-deficient B cell progenitors to the same extent as it did in wild-type control cells. Moreover, disruption of conventional Akt/TSC/Rheb signaling axis did not affect IL-7-controlled mTORC1 activation or early B cell development. Taken together, our current study for the first time demonstrates that the PLCγ pathway is critical for cytokine-mediated biological function, such as IL-7R-regulated early B lymphopoiesis, and this pathway directs IL-7R-mediated early B cell development through a novel and thitherto-unknown PLCγ/DAG/PKC-dependent but not conventional Akt/Rheb-dependent activation of mTORC1.